[Histonet] RE: IF staining on FFPE tissue

[Merced M Leiker]

Don't know if anyone else replied to this...but maybe try tinkering with 
your angtigen retrieval? Like try altering the temp, duration, or even the 
pH?  pH can be finicky for some epitopes, I've found...

Regards,
Merced

--On Wednesday, December 23, 2009 12:56 PM -0600 "Margaryan, Naira" 
<NMargaryan <@t> childrensmemorial.org> wrote:

> Dear Histonetters,
>
> Hopefully you are enjoying a great Holidays with your friends and family!
>
> I have been asked to perform . I got positive and negative absolutely
> identical with beautiful nuclear DAPI and very red RBC. What to do or
> what to change in my protocol to avoid RBC and background? Here is my
> protocol: 1.Deparaffinization  (xyline- 60min, 100%Eth- 15min, 95%- 5min,
> 70%- 5min, H2O) 2. Antigen Retrieval ph6 in Citrate buffer (same I use
> for IHC)
> 3. Wash H2O and TBST
> 4. Protein block- 10min
> 5. Ab, Rb anti-human - 60-90 min (same I use for IHC)
> 6. Wash TBST -5-10min
> 7. secondary Donkey anti-Rb 594 red - 60min
> 8. Wash TBST -5-10min, H2O -5min
> 9. DAPI - 10min
> 10. Wash H2O -5-10min
> 11. Gelvatol coverslip
>
> Am I using wrong secondary? Is there any specific secondary or protocol
> for IF staining for FFPE  to avoid background? I just used same AR and
> primary Ab's dilution like i use for IHC with nice results.
>
> Any suggestions are appreciated, especially in these Holidays days from
> working histo- people:)
>
> Thanks and have a warm and nice Holidays,
> Naira
>
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Merced M Leiker
Research Technician III
Cardiovascular Medicine
348 Biomedical Research Building
State University of New York at Buffalo
3435 Main St, Buffalo, NY 14214  USA
leiker <@t> buffalo.edu
716-829-6118 (Ph)
716-829-2665 (Fx)

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